Figure 6.

Two-Temperature LATE-PCR endpoint genotyping of the rs2270517 SNP in blinded DNA samples. Control replicate DNA samples corresponding to various known genotypes for the rs2270517 SNP were analyzed by Two-Temperature LATE-PCR using primer/probe pairs for the rs2270517 (C/T alleles) and a mismatch-tolerant probe specific for the C allele at high temperatures. Fluorescence signals were collected after LATE-PCR amplification at 57°C and 45°C and were used to calculate fluorescence ratios. In an initial experiment, replicate samples of each rs2270517 genotype (n = 26) were used to determine the distribution of fluorescence ratios corresponding to each genotype. Red dots indicate homozygous CC control samples, blue dots indicate the heterozygous CT control samples, and green dots indicate homozygous TT control samples. The boxes represent the boundary of three-tandard deviations on either side of the mean of replicate control samples signals for each genotype. Therefore, each box defines the range of fluorescence ratios statistically encompassing 99.7% of all possible samples of any given genotype in this assay. In two subsequent separate experiments, samples of known rs2270517 genotype that were blinded to the experimenter (coded samples A, B, C, D, E, and F in the figure) along with a set of control samples of known genotypes were processed to determine their fluorescence ratios in duplicate. These fluorescence ratios are shown in the figure as orange circles labelled with the sample designation; some of the circles from individual samples overlap in the figure. The data shows that the fluorescence ratios from each of the blinded samples fell within one of the defined ranges of normalized control fluorescence ratios. The same was observed for the set of control samples (shown as red, blue, or green dots according to genotype as specified above). There was 100% concordance in the genotype assignment when the blinded samples were decoded.